Project Summary Changes in cell fate ultimately occur through the acquisition of cell type-specific gene expression programs that are enabled by cooperation between the chromatin landscape and transcription factor availability. The deposition and removal of the chemical modifications that decorate chromatin require metabolites that are intermediates of metabolic pathways, while several enzymes that remove these marks use metabolites as part of their enzymatic reaction. Thus, cellular metabolic activity can shape gene expression programs through metabolite-dependent effects on chromatin organization. A robust gene regulatory network and permissive chromatin landscape are hallmarks of the nave pluripotent state in embryonic stem cells (ESCs), yet how intracellular metabolic pathways contribute to the establishment of this distinct chromatin landscape remains unclear. Our previous work demonstrated that nave mouse ESCs in the ground state of pluripotency alter their metabolic flux to support larger intracellular pools of the metabolite alpha-ketoglutarate (?KG) compared to their more committed counterparts. Supplementation of more committed ESCs with exogenous, cell-permeable ?KG is sufficient to increase self-renewal. However, how nave ESCs rewire metabolic pathways to promote ?KG accumulation, and how ?KG enhances self-renewal, remain open questions. The aim of this research proposal is to identify the pathways that support ?KG accumulation and determine the mechanism by which ?KG promotes self-renewal. The PI3K/Akt signaling axis is a well- known regulator of cellular metabolism and has been shown to support ESC self-renewal. Whether this signaling axis plays a role in ESC metabolism, particularly ?KG regulation, remains unexplored. Using mass spectrometric analysis combined with pharmacologic and genetic approaches, we will test the hypothesis that increased glucose oxidation mediated by Akt signaling is a major driver of the ?KG accumulation observed in nave ESCs. Given that ?KG serves as an obligate co-substrate for multiple enzymes that catalyze the removal of DNA methylation and repressive histone marks, we hypothesize that ?KG accumulation drives loss of repressive chromatin marks at the locus of Nanog, a core pluripotency transcription factor, thereby driving increased Nanog expression and stabilization of the pluripotency-associated gene regulatory network. We will use genetic and pharmacologic approaches to determine whether ?KG accumulation stimulates self-renewal by enhancing Nanog expression through a chromatin-mediated mechanism. These studies will address how mouse ESCs couple metabolic pathways with regulation of the pluripotency gene regulatory network and will provide critical insight into how metabolic regulation contributes to changes in cell identity.